The invention relates to a microplate with transparent bottom as well as to a method for its manufacture.
Microplates are known which are used for fluorescence, luminescence or scintillation measurements, for example, in solving biochemical or molecular biological questions.
New luminescence and fluorescence techniques require the preparation of colored microplates with transparent bottoms. Microplates with 96 wells today represent a standardized platform for automatic or manual determinations, and for evaluation of patient specimens in widespread analysis equipment. A routine method for the preparation of colored microplates with transparent bottoms is ultrasound welding of a colored plate frame to a transparent bottom. It is preferable that both parts be made of polystyrene. Achieving an absolute seal between the 96 wells always turns out to be a problem. Therefore, double welding beads are frequently applied to achieve a greater level of safety.
EP-0,571,661 A1 discloses a microplate which is used in measurement techniques where the light emission or light permeability is measured. The disclosed microplate compresses a cuvette-forming upper light-impermeable frame part as well as a light-permeable bottom part, which is attached by means of ultrasound welding to the upper frame part. There are also known variants of these microplates in which, below the transparent bottom part, a protective grid is applied which is made of nontransparent material, and which leaves clear the optical window. Manufacture of such microplates may employ multicomponent injection molding processes, where, by means of two injection molds, the frame and bottom parts are manufactured and assembled.
The drawback of the known microplates is that the transparent bottom parts, because of their thickness of approximately 1 mm, present light conduction effects which are the result of the deflection and total reflection of light. Total reflection occurs whenever light moves through an interface between an optically denser medium and an optically less dense medium, and the limiting angle, which is specific for the material, is exceeded. This property is used to good effect today in light conduction technology. Light is fed into one end of a light conductor, then exits through the other end, practically unweakened because of the total reflection. To achieve this effect, the walls of the fibers must, however, be absolutely smooth at optical dimensions. If this is not the case, as in injection molded part, then the reflection of the light is not total, only partial, and therefore it can exit through adjacent walls, or cuvettes. The adverse light conduction effect also occurs, for example, during transmitted light measurements, and its characteristics include the fact that the transparent bottom acts as a light conductor and, for given cuvettes, deflects incident light partially into adjacent cuvettes. In this context, it has been observed that, as the thickness of the bottom increases, the light conduction effect also increases, that is, the measurement accuracy decreases. In addition, the known microplates are only suited under certain conditions for radioactivity measurements, for example, scintillation measurements, also because of the thickness of their transparent bottoms.
The technical problem which is the foundation of the present invention thus consists in preparing microplates which overcome the above-mentioned drawbacks, particularly which guarantee a higher accuracy during optical measurements and, moreover, are also suited for radioactive determinations.
The invention solves this problem by the preparation of a microplate with the characteristics of the main claim, particularly by the preparation of a microplate with at least one frame part and at least one bottom part associated with the frame part, where the frame part, of which there is at least one, comprises multiple cuvettes and the bottom part, of which there is at least one, forms the bottoms of the cuvettes, and where the bottom part, respectively the bottoms, of the cuvettes has/have a maximum thickness of 500 xcexcm, preferably 20-500 xcexcm, and optimally 40 to 100 xcexcm.
In connection with the present invention, the term frame part of a microplate is understood to refer to that part of a microplate which forms the cuvettes or recesses, in particular their lateral walls, which are open towards both the top and bottom. The term bottom part of a microplate is understood to refer to that part of a microplate which closes off the cuvettes, and optionally the cuvette interstices, towards the bottom.
In connection with the present invention, the term cuvette is understood to refer to a container which can be made of any material, preferably plastic, and which is in the form of a cupule, well, bore, hollowed-out part or similar configuration, and which is used for receiving the samples the be examined. In a particularly preferred manner, the entire bottom part, or only those parts of the bottom part which form the bottoms of the cuvette, are made in the form of a membrane or, particularly, a transparent film.
The invention thus makes available, in an advantageous manner, a microplate which provides, because of the very small thickness of the bottom part, respectively the bottoms of the cuvettes, multiple advantages and applications. Because of the small thickness of the bottom part, respectively the bottoms of the individual cuvettes, it is, for example, possible and particularly advantageous for use in radioactivity determinations. To the extent that the bottom part is in the form of a transparent film, the resulting advantage is that the undesired light conduction effect is considerably reduced, so that measurements can be carried out with considerably increased accuracy, compared to the state of the art. To the extent that the bottom part is in the form of a membrane, it is possible to carry out any desired nutrient diffusion from the bottom through the membrane into the cells that grow on the membrane in the cuvette in a particularly efficient manner, and to a large extent unimpeded.
The microplates according to the invention are therefore suited for any kind of fluorescence, luminescence, colorimetric, chemilumescence or radioactivity measurement, for example, scintillation measurements. The microplates according to the invention can be used in ELISA tests, DNA and RNA hybridizations, antibody titer determinations, protein, peptide and immuno tests, PCR and recombinant DNA techniques, hormone and receptor binding tests and similar techniques. Since it is preferable to use, for the bottom part, respectively the bottoms of the individual cuvettes, transparent, that is light-permeable, materials, optical apparatuses can be placed both above and below the microplate. In addition it is possible to study the samples contained in the cuvettes using light microscopy.
The microplate according to the invention presents at least a frame part and at least one bottom part associated with the frame part. The frame part, of which there is at least one, is preferably essentially in the form of a rectangle and it comprises multiple cuvettes which are open towards the top and towards the bottom, in a support plate, where the lateral walls of the cuvettes are formed by the frame part which in this area is in the form of a support plate. The cuvettes that are formed in the frame part can have a circular, hexagonal, square or other geometric cross section. The cuvettes are arranged in the support plate of the frame part in the form of a matrix or in rows. The cuvettes in the support plate can be in the form of distinct individual cuvette, connected, for example, by connecting rods, in the form of bores in an otherwise solid support plate, or in the form of a combination of the two embodiments. Per frame part it is possible to have 6, 12, 24, 48, 96 or multiples thereof, for example, 384 or 1536, cuvettes. The frame part is applied in the injection molding process onto the bottom part which has a maximum thickness of 500 xcexcm, which bottom part thus closes the cuvettes from the bottom and at the same time forms the bottom for each individual cuvette. The microplate according to the invention can consist, for example, of such a frame part and a bottom part associated with this frame part. However, it is also possible according to the invention to use a design where one or preferably more frame parts are arranged in the middle of a foundation frame, in a manner so that it/they can be removed. Such a microplate accordingly comprises a foundation frame and frame parts, each provided with a bottom part, which are arranged in the foundation frame.
In another preferred embodiment, the invention provides for the coloring of the frame part, either black or white, or in natural, transparent, colors. In a particularly preferred manner, the frame part is manufactured of one material type or of a mixture of materials, guaranteeing an increased heat conductivity, for example, by the inclusion of metal shavings, such as shavings of nickel, special steel, or rust.
In a particularly advantageous embodiment, the invention provides for manufacturing the frame part of acrylbutadienestyrene (ABS), polyamide (PA), polycarbonate (PC), polystryene (PS), polymethyl methacrylate (PMMA), polypropylene (PP) or styrene acrylonitrile (SAN).
In an additional advantageous embodiment, the invention provides for manufacturing the film of a transparent material, or for coloring it. According to the invention it is possible to use a film with increased heat conductivity, for example, by using aluminum as film material. In a particularly preferred embodiment of the present invention, the film is constructed in several layers, where, for example, the face of the film turned toward the frame part is chosen so that it provides for a particularly good connection to the frame part, whereas the face of the film turned away from the frame part is used to improve the stability.
It is particularly preferred for the film to be made of acrylobutadienestyrene (ABS), polyamide (PA), polycarbonate (PC), polystryene (PS), polymethyl methacrylate (PMMA), polypropylene (PP) or styrene acrylonitrile (SAN), and for it to consist of these materials or mixtures thereof.
In a particularly preferred embodiment, the membranes according to the invention are manufactured of the polyamide (PA6, PA66), polyester (PET, PETG), polycarbonate (PC), cellulose, cellulose derivative or regenerated cellulose, and they consist of these materials or mixtures thereof.
Preferably the invention provides for a constant thickness of the bottom part and for the bottom part to be made in its entirety of the same material. However, the invention also provides for the bottom part to have a maximum thickness of 500 xcexcm only in those areas in which the bottom part forms the bottom of the respective cuvette, whereas in the areas between the cuvette bottoms and/or in the areas below the cuvette lateral walls a larger thickness of the bottom part is provided and/or another material composition.
The invention, in an additional advantageous embodiment, provides for a support structure below the bottom part which is used for stabilizing the bottom part and which is attached by welding or applied by spraying onto the bottom part or onto the frame part itself. Advantageously, this support structure leaves an optical window free, in each case under the cuvette bottom.
Furthermore, the invention provides for a notching or other marking of at least one, preferably two, corners of the frame part or of the foundation frame, so that an equivocal orientation can be used.
The invention also relates to another method for the manufacture of microplates consisting of at least one frame part and at least one bottom part, where the bottom part has a maximum thickness of 500 xcexcm. The invention provides for the manufacture of the microplates according to the invention in a single-step procedure, where the bottom part, which is in the form of a film or membrane, is arranged in an injection molding installation and subsequently a molding composition, heated at 200-300xc2x0 C., preferably 250xc2x0 C., and plasticized, is injected for the manufacture of the frame part into the injection molding installation and applied to the bottom part.
The method according to the invention provides for a thin, prestamped, film or membrane, having preferably a thickness of 60 xcexcm, which is inserted into an injection molding mold, and around which is sprayed the material used for the frame part and connected to it. The material can be transparent, or colored with a white or black with high covering power. The fixation of the film or membrane can be carried out using a vacuum through small channel slits, which, however, do not leave any visible imprints on the mold part, or by electrostatic charging of the film, or the membrane and/or injection molding tool.
Using this method, it is possible, for example, to use the application process by spraying around films or membranes made of polymethyl methacrylate, polyester, or polycarbonate, and having a thickness range of 20-500 xcexcm. According to the invention, it should be considered preferable to obtain a good connection between the film or membrane and the mold composition sprayed around it. Optionally, the film or membrane according to the invention can be subjected to a preliminary treatment using the corona or plasma processes respectively, preactivated with appropriate adhesives. The temperature resistance of the composite is a function of the film or membrane and of the mold composition used for the manufacture of the frame part and it is, for example, in the case of polystyrene approximately 50xc2x0 C. It is preferable to ensure that no thermal deformation of the film or membrane occurs, when the heated mold composition (approximately 250xc2x0 C.) is injected. The injection molding tool should be designed in such a manner that the film or membrane is not shifted.
Using appropriate films or membranes, it is possible to use this processing technique to achieve a great number of different objectives. The objectives include improved properties of use, such as high light permeability and good resistance to chemicals. Furthermore, according to the invention it is possible to render the film surfaces hydrophilic or hydrophobic by plasma or corona processes, and to incorporate functional amino groups. Microplates modified according to the invention can be used in immunoassays and cell cultivation. According to the invention, it is also possible to apply sprays around the membranes which are to be used for cell culture techniques and filtration processes.
A sterilization which may be required with accelerated electrons or gamma quanta does not lead to any noteworthy changes in the materials.
The use of an adapted tool makes it possible to manufacture in small or large series with a good cost-benefit ratio. Without long interruption or production, it is thus possible to manufacture different qualities of films or surfaces.
Other advantageous embodiments of the invention can be obtained from the secondary claims.